Vitamin K dependent (VKD) proteins require carbolylation for activity and so the VKD carboxylase plays a critical role in hemostasis. The carboxylase converts Glu's to Gla's using the energy of vitamin K oxygenation. At least 10 different VKD proteins in liver engage a single carboxylase in the endoplasmic reticulum to become fully carboxylated (9-12 Gla's per molecule). Under normal conditions full carboxylation occurs, however how the process is regulated to achieve such remarkable fidelity is not known. A method that mimics the normal physiological process was developed to study protein carboxylation, which showed conclusively that carboxylase processivity can account for comprehensive carboxylation in vivo and also provided new insights into the mechanism. The carboxylase active site was identified in studies that led to a hypothesis of substrate-regulated carboxylation. The long standing question of why carboxylation is saturated when VKD proteins are expressed in mammalian cells was addressed and showed the importance of the secretory machinery in facilitating carboxylation. Two modifications, carboxylase carboxylation and phosphorylation, were discovered. The long term goal is to understand the mechanism of carboxylation, including how multiple VKD proteins compete for one carboxylase in tissue to become fully carboxylated. The specific aims proposed for the next funding period are to: 1) Determine how the active site facilitates carboxylation. The hypotheses for how carboxylation is regulated will be tested by characterizing a newly discovered second site of VKD protein-carboxylase interaction and determining how vitamin K is oxygenated to initiate carboxylation. 2) Define the mechanism of VKD protein carboxylation, The hypothesis that vitamin K availability, competing VKD proteins and carboxylase carboxylation affect carboxylase processivity will be tested. 3) Determine how carboxylase carboxylation and phosphorylation regulate carboxylation. The hypothesis that these modifications regulate how secretory events facilitate carboxylation will be tested by analyzing effects on carboxylase trafficking, stability, chaperone association and fIX carboxylation. These studies will make fundamental contributions towards understanding the mechanism of carboxylation, which will be important for developing improved anticoagulants, determining how changes in diet and anticoagulation affect hemostasis and producing therapeutic amounts of VKD proteins.